1. Volume 10, Issue 3, Pages 203-214 (September 2006)
C/EBPβ at the core of the TGFβ cytostatic response and its evasion in metastatic breast cancer cells 
Roger R. Gomis, Claudio Alarcón, Cristina Nadal, Catherine Van Poznak, Joan Massagué 
Cancer Cell 
Volume 10, Issue 3, Pages (September 2006)
DOI: /j.ccr
Copyright © 2006 Elsevier Inc. Terms and Conditions

2. Figure 1 Smad, FoxO, and C/EBPβ transactivate p15INK4b
A: Graphic comparison of the p15INK4b promoter with the p21CIP1 promoter and the dual TGFβ input. The nucleotide sequence of the human and mouse p15INK4b Smad binding regions (SBR1 and SBR2) containing binding elements for Smad (SBE, orange), Forkhead (FHBE, green), and C/EBPβ (C/EBPβBE, blue) are shown.
B: HaCaT cells were transfected with transcriptional reporter construct based on the −751/+70 p15INK4b promoter region driving firefly luciferase expression. Where indicated, the cells were cotransfected with siRNA against FoxOs, or with vectors encoding FoxO3, C/EBPβ LAP2, or Smad3. Cells were then treated with TGFβ for 20 hr, and luciferase activity was determined. Luciferase activity was normalized based on a cotransfected CMV-renilla luciferase vector. Data are mean ± SD (n = 3).
C–E: HaCaT cells transfected with luciferase reporter constructs driven by 4× multimers of p15SBR1, p15SBR2, and p21SBR (C), 4× multimers of p15SBR2 and the indicated mutations (D), or 4× multimers of p15SBR2 (E). FoxO, C/EBPβ, or Smad expression vectors were cotransfected with the reporter constructs as indicated. Cells were then treated as in B.
Cancer Cell  , DOI: ( /j.ccr )
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3. Figure 2
TGFβ-inducible binding of Smad, FoxO, and C/EBPβ to the p15INK4b promoter
A: A schematic representation of the human p15INK4b promoter regions amplified in ChIP assays. The SBEs (orange), FHBE (green), C/EBPβBE (blue), and INR (gray) are indicated.
B: HaCaT cells were incubated with or without TGFβ for 90 min and subjected to ChIP assays with the indicated antibodies and PCR primers. The proximal region of the p15INK4b promoter was used as a negative control.
C: HaCaT cells were transfected with the indicated Flag-tagged expression vectors and cultured for 24 hr. Cells were then incubated with or without TGFβ for 90 min and subjected to ChIP assays with anti-Flag antibodies and the indicated PCR primers.
D: HaCaT cells were treated with or without TGFβ for 90 min, and lysates were immunoprecipitated with the indicated antibodies and then subjected to immunoblotting with anti-Smad4 antibody.
Cancer Cell  , DOI: ( /j.ccr )
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4. Figure 3 Requirement of C/EBPβ for cytostatic TGFβ gene responses
A: HaCaT cells infected with a retroviral vector encoding the LIP isoform of C/EBPβ or an empty retrovirus vector were incubated in the presence or absence of TGFβ for 3 hr. Total RNA was subjected to Northern blot analysis using the indicated probes. Whole-cell extracts from cells incubated with or without TGFβ for 5 hr were subjected to immunoblotting with the indicated antibodies.
B: Total RNA from control and C/EBPβ-depleted HaCaT cells was subjected to Northern blot analysis using the indicated probes.
C: Immunoblots with the indicated antibodies were performed on whole-cell extracts from control and C/EBPβ-depleted HaCaT cells.
D: Control and C/EBPβ-depleted HaCaT cells were treated with or without TGFβ for 3 hr. Total RNA was subjected to Northern blot analysis using the indicated probes. Whole-cell extracts were treated as in A.
Cancer Cell  , DOI: ( /j.ccr )
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5. Figure 4 Requirement of C/EBPβ for the TGFβ growth-inhibitory response
A: HaCaT cells transfected with a firefly luciferase reporter construct driven by the TGFβ-responsive region of the c-Myc promoter plus a LIP expression vector as indicated were treated with TGFβ for 20 hr, and luciferase activity was determined. TGFβ-responsive luciferase activity was normalized against the activity of cotransfected renilla luciferase driven by a constitutive promoter. Data are mean ± SD (n = 3).
B: HaCaT cells were incubated with or without TGFβ for 90 min and subjected to ChIP assays with the indicated antibodies and PCR primers for the proximal region of the myc promoter. The β-ACTIN promoter was used as a negative control.
C and D: Growth curves of control and Lip-overexpressing HaCaT cells (C) or control and C/EBPβ-depleted HaCaT cells (D) with or without TGFβ. Data are average of triplicated cell counts ± SD.
Cancer Cell  , DOI: ( /j.ccr )
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6. Figure 5
Evasion of TGFβ cytostatic responses in metastatic breast cancer cells is linked to LIP/LAP imbalance
A: EpCAM+ cells sorted from pleural effusion fluids from breast cancer patients were cultured in the presence or absence of TGFβ for 3 hr. Total RNA was subjected to Northern blot analysis using the indicated probes. Whole-cell extracts were probed with the indicated antibodies. Bottom: cells were treated for 20 hr with the indicated concentrations of TGFβ and pulsed for 6 hr with 125I-deoxyuridine. 125I-dU incorporation was determined, and the data are plotted as percentage incorporation relative to control cells that received no TGFβ. Data are mean ± SD (n = 3).
B: Whole-cell extracts from the cell samples described in A were probed with the indicated antibodies.
C: The relative inhibition of 125I-dU incorporation by 100 pM TGFβ is plotted against the LIP:LAP molar ratio in each cell sample described in B.
Cancer Cell  , DOI: ( /j.ccr )
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7. Figure 6 TGFβ responses in breast cancer cells
Expression levels of different TGFβ target genes in six primary breast cancer cell samples that were incubated with or without TGFβ for 3 hr and analyzed by qRT-PCR using primers for the indicated genes.
Cancer Cell  , DOI: ( /j.ccr )
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8. Figure 7
Lap restores TGFβ cytostatic responses in resistant breast cancer cells
A: Tumor cell samples CN37 and CN41 were infected with a retroviral vector encoding the LAP2 C/EBPβ isoform or a GFP fusion protein as control. Transduced cells were selected for 48 hr based on a puromycin resistance marker, allowed to recover for another 24 hr, and then treated with or without TGFβ for 3 hr. Total RNA was subjected to Northern blot analysis using the indicated probes. Whole-cell extracts were probed with the indicated antibodies. c-MYC levels were normalized against β-ACTIN and were used to calculate the TGFβ fold repression. Data are mean ± SD of three independent experiments.
B: Total RNA from sample CN37 in A was subjected to qRT-PCR analysis using the indicated probes. Data are mean ± SD of three independent experiments. ND, not detectable signal.
C: CN37 and CN41 cells treated as in A were then incubated for 20 hr with the indicated concentrations of TGFβ and cultured in the presence of 125I-dU for an additional 6 hr. 125I-dU incorporation into DNA was determined. Data are mean ± SD (n = 3). The 125I-dU incorporation of the 100 pM TGFβ condition compared to controls that did not receive TGFβ is indicated for each cell sample.
D: Growth in mammary fat pad (MFP) of CN37 cells stably transfected with a retroviral construct encoding firefly luciferase and also stably transfected with control or LAP2 vector. Each curve shows tumor luminescence as a normalized photon flux emission. The mice images are representative of each group.
E: Schematic model of a set of cytostatic gene responses triggered by TGFβ in keratinocytes and primary breast cancer cells. Induction of p21CIP1 and p15INK4b are mediated a by FoxO-Smad combination, whereas repression of c-MYC is mediated by a E2F4/5-Smad combination. The p15INK4b and c-MYC responses are enabled by the active C/EBPβ isoform LAP2, which is opposed by the natural dominant-negative inhibitory isoform LIP. In metastatic breast cancer cells with a high LIP:LAP ratio, the p15INK4b and c-MYC are cancelled. See the text for details and references.
Cancer Cell  , DOI: ( /j.ccr )
Copyright © 2006 Elsevier Inc. Terms and Conditions